Abstract Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea in hospitalized patients, with an estimated annual cost to the United States of between $750 million and $3.2 billion. The pathology of CDI is caused by the toxins secreted by the bacteria. Recently, two anti-C. difficile toxin antibodies, actoxumab and bezloxumab, that bind to and neutralize C. difficile toxins A and B, respectively, have achieved success for CDI treatment in a phase II clinical trial. However, only bezloxumab demonstrated efficacy in the subsequent phase III clinical trial. The neutralization potency of antibody is strongly epitope-dependent. Since both actoxumab and bezlotoxumab were engineered using toxins from laboratory C. difficile strain VPI 10463, both showed significantly reduced neutralization potencies against some recently emerged hypervirulent strains of C. difficile. Thus, there is an urgent need to develop broadly effective C. difficile toxins neutralizer. We propose to engineer a non-antibody protein, designed ankyrin repeat protein (DARPin), that is able to bind to and neutralize toxins A and B from a broad range of C. difficile strains. DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity. Furthermore, DARPin can be very efficiently expressed in E. coli (accounting for >50% of all E. coli proteins) and very easily purified due to its high thermostability. In this project, we will first use bacteriophage display to isolate DARPins that are able to bind to toxins from different strains of the C. difficile (Aim 1) and then subject the selected DARPins to an anti-toxin functional screen in cultured cells to identify toxin-neutralizing DARPins (Aim 2). The potency of the selected DARPins against a panel of emerging and clinically relevant strains of C. difficile will be analyzed and be further optimized by directed evolution (Aim 3). Successful completion of this study will yield an arsenal of high-potency toxin-neutralizing DARPins. These broadly neutralizing anti-toxin DARPins can potentially be fused to Fc and used as antibody therapy for CDI, or be formulated for oral administration to directly neutralize C. difficile toxin(s) in the gut. The approach of neutralizing bacterial virulence factors with DARPins should also offer a new treatment paradigm for other bacterial infection.